ALCOHOL METABOLISM GENES IN TRANSGENIC MICE

描述（由申请人提供）：酒精引起的肝损伤和疾病是一个重要的健康问题，肝脏中至少有三种酶活性能够将乙醇转化为乙醛：酒精脱氢酶（ADH）、细胞色素P450 2E1 （CYP2E1）和过氧化氢酶。尽管慢性酒精暴露可诱导CYP2E1，但使用自然发生和诱导的ADH缺陷动物的结果表明，I类ADH在乙醇代谢中起主要作用，IV类ADH起重要作用。一类ADH在哺乳动物的肝脏中有高水平表达，在小鼠中有一个单一基因Adh1编码一类ADH，而在人类中有三个基因ADH1A、ADH1B和ADH1C编码一类多肽。并通过基因拷贝数对肝脏中ADH1/CYP2E1的比值进行遗传操纵，探讨该比值在酒精性肝损伤中的作用。BAC克隆的远端顺式连锁序列将指导肝脏中ADH1和胃中Adh4的表达，目前还没有任何人类I类ADH基因指导肝脏表达的序列。遗传学方法将确定Adh1基因的重要远端控制元件，并在恒定的C57BL/6背景下构建和使用不同Adh1 /CYP2E1比例的小鼠菌株，以探索它们在酒精性肝损伤中的作用。具体目的是：(1)通过转基因表达试验，实验地描绘控制Adh1在肝脏、肠道和肾上腺中表达的远端序列的位置；(2)确定是否在表达的Adh1区域发生染色质改变，(3)一般定义促进Adh4适当表达表型的顺式连锁序列，(4)通过基因敲除和转基因构建Adh1 /CYP2E1比例变化8倍的小鼠品系，(b)在肝脏缺乏ADH的情况下，在肺、睾丸、精囊和附睾中过表达ADH（“条件敲除”），(c)携带人类CYP2E1 BAC进行过表达。(5)探讨乙醇对这些遗传菌株的促进作用。

DESCRIPTION (provided by applicant): Alcohol-induced liver damage and disease is an important health problem At least three enzyme activities in liver are capable of converting ethanol to acetaldehyde, alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1), and catalase Although CYP2E1 is inducible by chronic alcohol exposure, results using naturally occurring and induced ADH-deficient animals suggest class I ADH plays a major role in ethanol metabolism and class IV plays a significant role. Class I ADH is expressed at high levels in the liver of mammals A single gene, Adh1, encodes class I ADH in mice while three genes, ADH1A, ADH1B and ADH1C, encode class I polypeptides in humans The overall goals are to identify distal genomic dements that control the expression phenotype of members of the mouse Adh complex, and to genetically manipulate by gene copy-number the ratio of ADH1/CYP2E1 in liver to explore the role of this ratio in alcohol-induced liver damage Distal cis-linked sequence in a BAC clone will direct expression of Adh1 in liver and Adh4 in stomach No sequence directing liver expression of any human class I ADH gene is known. Genetic approaches will define the important distal control element(s) for the Adh1 gene, and construct and use mouse strains on a constant C57BL/6 background with varying ADH1/CYP2E1 ratios to explore the role of each in alcohol induced liver injury The specific aims are (1) experimentally delineate the location of the distal sequence(s) controlling expression of Adh1 in liver, intestine and adrenal using transgenic expression assays, (2) determine if chromatin alterations occur in the expressed Adh1 region, (3) generally define the cis-linked sequence that promotes Adh4 proper expression phenotype, (4) genetically construct mouse strains (a) having ADH1/CYP2E1 ratio varying 8- fold using available knockouts and transgenics, (b) overexpressing ADH in lung, testes, seminal vesicle, and epididiymis in the absence of ADH in liver (a "conditional knockout"), (c) harboring a human CYP2E1 BAC for overexpression, and (5) explore pathology promoted by ethanol in these genetic strains.